The field of the invention disclosed herein relates generally to vectors and plasmids that can replicate in a broad range of gram-negative host bacterial species. There have been several broad host range plasmids, described in the prior art, capable of being transmitted by bacterial conjugation and capable of replicating in a broad range of gram-negative bacteria. Furthermore, such plasmids have been modified to improve their suitability as cloning vehicles in gram-negative strains. See, e.g., Bagdasarian, M., et al., in "Plasmids of Medical Environmental and Commercial Importance" (K. N. Timmis and A. Puhler, eds.), Elsevier, Neth. Holland (1979) p. 411; Bagdasarian, M., et al., Gene 16, 237 (1981); and Bagdasarian, M., et al., in "Microbial Drug Resistance, Proc. 3rd Intl. Symp. Tokyo" (S. Mitsuhasi, Ed.) (1982) p183 and Ditta, G., et al., Proc. Nat. Acad. Sci. USA 77, 7347 (1980). Examples include RSF1010, a natural isolate, which belongs to incompatibility group Q and is mobilizable by plasmids of incompatibility groups P or Q, but not self-transmissable. (For a general discussion of incompatibility groups, see Datta, N. "Plasmids of Medical, Environmental and Commercial Importance", ibid. page 3.) The behavior of such vectors stands in sharp contrast with the plasmids commonly used for genetic engineering in E. coli. Such plasmids display a very narrow host range within which they are able to replicate, despite the fact that E. coli is itself a gram-negative organism. In the case of the commonly used E. coli vectors, their replication is confined to E. coli and closely related species, such as Salmonella. Their usefulness is therefore confined to genetic manipulations in E. coli and its close relatives (hereinafter referred to as the E. coli group). Genetic enginnering and recombinant DNA work outside the E. coli group has heretofore been severely limited by the availability of plasmids having useful features, such as drug-resistance markers, a variety of well-placed insertion sites, and the like. Modifications of the natural broad host range plasmid RSF1010 have provided some improvements, such as the plasmid pKT210, see Bagdasarian, M. et al., in "Plasmids of Medical, Environmental and Commercial Importance", supra page 411. Plasmid pKT210 has two drug-resistance genes, for chloramphenicol and for streptomycin together with two potential insertion sites, HindIII and EcoRI. The general usefulness of pKT210 is limited somewhat by the nature of the drug-resistance markers. For example, the chloramphenicol resistance gene is poorly expressed in Rhizobium meliloti and further, one of the more commonly used strains of R. meliloti, strain 2011, already carries a streptomycin resistance marker. Plasmids RSF1010 and pKT210 cannot be used for cosmid cloning since they lack a cos site. A difficulty encountered with prior art vectors that contain a cos site has been plasmid instability when the plasmid is used to carry large inserts in the range of 30-40kbp such as normally used for cosmid cloning.
It would therefore be highly advantageous to obtain an improved cloning vector, that has retained the broad host range of plasmids such as RSF1010 and pKT210, while carrying improved genetic markers and a wider variety of useful insertion sites. Further, it would be highly desirable to obtain such a plasmid in a form that is stable when bearing a large DNA insert, such as are employed in cosmid cloning. Still further, such a vector should be capable of high copy number replication in host cells.